Stem and progenitor cells for liver repopulation: can we standardise the process from bench to bedside?

There has been recent progress in the isolation and characterisation of stem/progenitor cells that may differentiate towards the hepatic lineage. This has raised expectations that therapy of genetic or acquired liver disease might be possible by transplanting stem/progenitor cells or their liver-committed progeny. However, it is currently impossible to determine from the many documented studies which of the stem/progenitor cell populations are the best for therapy of a given disease. This is largely because of the great variability in methods used to characterise cells and their differentiation ability, variability in transplantation models and inconsistent methods to determine the effect of cell grafting in vivo. This manuscript represents a first proposal, created by a group of investigators ranging from basic biologists to clinical hepatologists. It aims to define standardised methods to assess stem/progenitor cells or their hepatic lineage-committed progeny that could be used for cell therapy in liver disease. Furthermore standardisation is suggested both for preclinical animal models to evaluate the ability of such cells to repopulate the liver functionally, and for the ongoing clinical trials using mature hepatocytes. Only when these measures have been put in place will the promise of stem/progenitor-derived hepatocyte-based therapies become reality.

Endothelial-like cells derived from human CD14 positive monocytes.

In the present study, we show that endothelial-like cells (ELCs) can develop from human CD14-positive mononuclear cells (CD14 cells) in the presence of angiogenic growth factors. The CD14 cells became loosely adherent within 24 h of culture and subsequently underwent a distinct process of morphological transformation to caudated or oval cells with eccentric nuclei. After 1 week in culture the cells showed a clear expression of endothelial cell markers, including von Willebrand factor (vWF), CD144 (VE-cadherin), CD105 (endoglin), acetylated low-density lipoprotein (AC-LDL)-receptor, CD36 (thrombospondin receptor), FLT-1, which is vascular endothelial cell growth factor (VEGF) receptor-1, and, to a weaker extent, KDR (VEGF receptor-2). Furthermore, in these cells structures resembling Weibel-Palade bodies at different storage stages were identified by electron microscopy, and upon culturing on three-dimensional fibrin gels the cells build network-like structures. In addition, cell proliferation and vWF expression was stimulated by VEGF, and the endothelial cell adhesion molecules CD54 (ICAM-1), and CD106 (VCAM-1) became transiently inducible by tumor necrosis factor-alpha (TNF-alpha). In contrast, the dendritic markers CD1a, and CD83 were not expressed to any significant extent. The expression of CD68, CD80 (B7-1), CD86 (B7-2), HLA-DR and CD36 may also suggest that ELCs might be related to macrophages, sinus lining or microvascular endothelial cells. Taken together, our observations indicate that ELCs can differentiate from cells of the monocytic lineage, suggesting a closer relationship between the monocyte/macrophage- and the endothelial cell systems than previously supposed.

In vitro differentiation of endothelial cells from AC133-positive progenitor cells.

Recent findings support the hypothesis that the CD34(+)-cell population in bone marrow and peripheral blood contains hematopoietic and endothelial progenitor and stem cells. In this study, we report that human AC133(+) cells from granulocyte colony-stimulating factor-mobilized peripheral blood have the capacity to differentiate into endothelial cells (ECs). When cultured in the presence of vascular endothelial growth factor (VEGF) and the novel cytokine stem cell growth factor (SCGF), AC133(+) progenitors generate both adherent and proliferating nonadherent cells. Phenotypic analysis of the cells within the adherent population reveals that the majority display endothelial features, including the expression of KDR, Tie-2, Ulex europaeus agglutinin-1, and von Willebrand factor. Electron microscopic studies of these cells show structures compatible with Weibel-Palade bodies that are found exclusively in vascular endothelium. AC133-derived nonadherent cells give rise to both hematopoietic and endothelial colonies in semisolid medium. On transfer to fresh liquid culture with VEGF and SCGF, nonadherent cells again produce an adherent and a nonadherent population. In mice with severe combined immunodeficiency, AC133-derived cells form new blood vessels in vivo when injected subcutaneously together with A549 lung cancer cells. These data indicate that the AC133(+)-cell population consists of progenitor and stem cells not only with hematopoietic potential but also with the capacity to differentiate into ECs. Whether these hematopoietic and endothelial progenitors develop from a common precursor, the hemangioblast will be studied at the single-cell level.

Derivation of a new hematopoietic cell line with endothelial features from a patient with transformed myeloproliferative syndrome: a case report.

BACKGROUND: During embryonal development primitive hematopoiesis can be observed first in the yolk sac, in which both hematopoietic and endothelial cells are derived from a common precursor, the hemangioblast. Whether cells with this dual differentiation potential persist during postnatal life is unknown. METHODS: A cell line was derived from a patient with secondary acute leukemia. Because of its ability to grow in soft agar and in SCID mice, this cell line was analyzed for expression of differentiation antigens by fluorescence-activated cell sorter analysis, immunocytochemistry, fluorescent in situ hybridization (FISH) analysis with simultaneous cell surface staining, and polymerase chain reaction (PCR). RESULTS: A new cell line was established from a patient with essential thrombocytosis that transformed into acute leukemia. The patient's initial clinical presentation included skin and lymph node infiltrations that were taken for an angiosarcoma due to positivity for CD34, CD31, and von Willebrand factor on immunohistology. In addition to hematopoietic markers, leukemic cells expressed endothelial antigens such as CD62E, CD105, and bound Ulex europäeus lectin-1. Immunocytochemistry revealed positive staining for vascular endothelial growth factor receptor type 2 (KDR), Tie-2/Tek, the angiopoietin receptor, and vascular endothelial cadherin. These results were confirmed by PCR analysis. Simultaneous staining for CD62E and FISH analysis showed that cells with endothelial characteristics belonged to the leukemia. FISH analysis of histologic sections of the lymph node infiltration confirmed this manifestation as part of the leukemic process. The derived cell line, UKE-1, forms colonies in soft agar and is tumorigenic in SCID mice. CONCLUSIONS: This new cell line, UKE-1, appears to combine hematopoietic and endothelial features, indicating the close ontogenic relation of both lineages.

Ex vivo expansion of megakaryocyte progenitors: effect of various growth factor combinations on CD34+ progenitor cells from bone marrow and G-CSF-mobilized peripheral blood.

Prolonged thrombocytopenia resulting from inadequate megakaryocyte (MK) progenitor cell reconstitution is a serious complication of hematopoietic cell-supported high-dose chemotherapy (HDC). In this situation, the infusion of MK progenitors that are expanded ex vivo could be clinically beneficial. In this study we investigated the ability of various growth factor combinations to generate MK progenitors. CD34+ cells derived from bone marrow (BM) and granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) from 17 patients with breast cancer, lymphoma, or myeloma were cultured unpertubed for 10 days in a serum-free liquid culture system that contained recombinant growth factors. Five different growth factors combinations were evaluated: Stem cell factor (SCF), interleukin (IL)-3, IL-6 + G-CSF (combination 1); SCF, megakaryocyte growth and development factor (MGDF) + G-CSF (combination 2); SCF + MGDF (combination 3); MGDF alone (combination 4); and SCF, IL-3, IL-6, G-CSF + MGDF (combination 5). PB CD34+ cells yielded significantly higher numbers of CD41+ MK progenitors than BM CD34+ cells with any of the growth factor regimens assayed. PB CD34+ cells (2x10[5]) at day 0 generated 1.2 to 1.3x10(6) CD41+ cells by day 10 when cultured in the presence of growth factor combinations 1, 2, or 3. In contrast, 2x10(5) BM CD34+ cells produced 5x10(5) CD41+ cells after 9 days in the presence of combination 1, whereas lower numbers of CD41+ cells were generated in cultures with combinations 2 and 3 (2.3x10[5] and 4.2x10[4], respectively). The addition of MGDF to cultures that were grown with combination 1 for 5 days increased the number of CD41+ cells (1.7-fold increase in PB-derived cultures, 1.6-fold increase in BM-derived cultures). Treatment with MGDF alone resulted in higher frequencies of MK progenitors than those obtained in cultures with combined growth factors (79% in PB-derived cultures, 25% in BM-derived cultures), but because total cell growth was attenuated, absolute numbers of MK progenitors were lower (7x10(5) in PB-derived cultures, 7x10(4) in BM). Morphological analysis of immunocytochemically identified megakaryocytic cells revealed mononuclear cells as the predominant cell type in all of the cultures. During the 10-day culture period, PB-derived MK progenitors did not show notable maturation, even under the influence of MGDF, whereas in BM-derived cultures MGDF induced a significant shift to binuclear cells and stage I MK after day 5. Phenotypic analysis of cell surface markers showed that the majority of cultured megakaryocytic cells coexpressed CD34 and platelet glycoproteins (GPs), also indicating an immature stage of development. The ex vivo proliferative activity of CD34+ cells and their potential to develop into the megakaryocytic lineage demonstrated considerably high interpatient variations. There was no correlation between platelet recovery following HDC with hematopoietic cell support and the magnitude of GP+ cell expansion ex vivo, suggesting the feasibilty of MK expansion ex vivo in patients with prolonged thrombocytopenia posttransplantation. In summary, these data indicate that GCSF-mobilized CD34+ PBPCs are more effectively expanded ex vivo into the megakaryocytic lineage than are CD34+ BMPCs. CD34+/GP+ MK progenitors may be an appropiate cell population for transplantion as prophylaxis or treatment of prolonged thrombocytopenia. The efficacy of this procedure will be tested prospectively in a clinical trial.

Bone marrow metastases.

This article discusses the clinical significance of bone marrow metastases and the current methods being used to detect tumor cells in marrow. The strategies being investigated for eradicating cancer cells from marrow in patients receiving hematopoietic cell autografts also are reviewed.

Stem cell isolation.

High-dose chemotherapy with autologous hematopoietic progenitor cell support is increasingly used to treat a variety of malignant diseases. A drawback of this technique is the potential for infusing clonogenic tumor cells with the autograft, producing relapse of the disease in the patient. The use of positive selection techniques to isolate stem cells and thus reduce or eliminate tumor cell contamination has been extensively studied over the past few years. Preliminary clinical results have demonstrated that these procedures deplete 2 to 7 logs of tumor cells and do not impair engraftment. It is too early to assess the ultimate clinical benefit of this strategy. Additional applications of CD34-selection include ex vivo expansion of and gene transfer into hematopoietic progenitor cells and T-cell depletion of allogeneic grafts to reduce the incidence of graft-versus-host disease.

[Mediastinal actinomycosis with formation of an esophagotracheal fistula. A case report]. / Mediastinale Aktinomykose mit ösophagotrachealer Fistelbildung. Ein Fallbericht.

Before antibiotics were available, actinomycosis was the most commonly diagnosed "fungal disease" of the lung because of its morphological similarity to true fungi. At that time actinomycosis presented a fairly typical clinical picture of empyema thoracis and sinus tracts in the chest wall. Nowadays it has become a rare infectious disease that is usually caused by the bacterium Actinomyces israelii and is amenable to treatment by most antibiotics available today. The following report describes the case of a 59-year-old man with an uncommon mediastinal actinomycosis that caused an oesophagotracheal fistula. This complication may develop due to the necrotizing inflammatory process that is typical for actinomycosis. With regard to the literature, the clinical manifestations of the disease and diagnostic and therapeutic considerations are discussed.

The pulmonary artery acceleration time determined with the MR-RACE-technique: comparison to pulmonary artery mean pressure in 12 patients.

The goal of our study was to evaluate a rapid noninvasive MR technique for quantification of the pulmonary artery acceleration time (PAT) and other parameters of pulmonary hemodynamics and to correlate with pulmonary artery mean pressure (mPAP). The PAT known as "time-to-peak" out of Doppler echocardiographic measurements normally shows significant inverse correlation with mPAP. With the MR-RACE-Technique (RACE: Real time ACquisition and Evaluation of motion) blood velocity measurements can be obtained with a total acquisition time of a few seconds. The application of this technique to the pulmonary artery has not been reported before. Out of the RACE velocity wave form PAT can be obtained with a temporal resolution of about 15 ms. To explore the relationship between PAT and mPAP, right heart catheterization and MR-RACE measurements were performed in 12 patients with different pulmonary vascular abnormalities. Results of MR-RACE were compared with those of mPAP measured by right heart catheter and showed significant inverse correlation (r = -0.82, p = .0011, n = 12). The ability of MR-RACE to enable measurements of blood flow with profiles may be important for characterizing pulmonary and cardiovascular abnormalities.

[Intravascular ultrasound in preoperative diagnosis of central bronchial tumors--initial clinical experiences]. / Intravasale Sonographie zur präoperativen Diagnostik zentraler Bronchialtumoren--erste klinische Erfahrungen.

In the course of preoperative diagnosis, intravasal sonography for tumour imaging was conducted in three patients suffering from central bronchial carcinoma. The catheters of 6.0 or 4.8 French diameter were advanced in each case after pulmonary angiography via the left or right pulmonary artery up to the tumour area. Endosonographic tumour imaging was compared with the findings of the other preoperative diagnostic measures and in two cases with intraoperative and postoperative findings. The vascular walls of the central pulmonary arterial segments showed sonographically no typical three-layer structure. In all cases, however, tumour infiltration was showed up by disappearance of the vascular wall reflexes in the relevant pulmonary artery branches. Visualisation of the mediastinal pulmonary artery segments or of the main stem of the pulmonary artery is difficult with the wire-guided catheters used, since these cannot be stabilised in the centre of the vessel. Development of suitable catheters with low-frequency transducers and greater depth of penetration is imperative especially for the diagnostically important visualisation of the surrounding mediastinal structures.

[Aortopulmonary fistula formation after operation of aortic isthmus stenosis as a rare cause of recurrent hemoptysis]. / Aortopulmonale Fistelbildung nach Operation einer Aortenisthmusstenose als seltene Ursache rezidivierender Hämoptysen.

An aneurysm of the aorta with aortobronchial fistula formation was seen in a 33 year old patient as a rare cause of recurring hemoptysis. 18 years previously he had undergone surgery of coarctation of the aorta. By means of different imaging methods the diagnosis was established in time and the aneurysm resected, so that the patient survived this mostly fatal incident.

[Measurement of the blood flow velocity in the pulmonary arteries using the magnetic resonance technique]. / Messung der Blutflussgeschwindigkeit in den Pulmonalarterien mit Hilfe der Magnetresonanztechnik.

MR blood velocity measurements were performed by the RACE technique in a plane perpendicular to the flow of the pulmonary arteries. MR findings were correlated with those of perfusion scintigraphy, Doppler US and right heart catheter (thermodilution). The ratio of MR blood flow measurements of right and left pulmonary arteries correlated well with the results of perfusion scintigraphy (RPA to LPA) and Doppler. Poor correlation was found when comparing MR blood flow measurements with right heart catheter since absolute flow measurements can be superimposed by neighboring blood vessels in complex anatomic situations.

[The echocardiographic demonstration of a right atrial thrombus and its embolization into the lung]. / Echokardiographischer Nachweis eines rechtsatrialen Thrombus und seiner Embolisation in die Lunge.

A 43-year-old man was hospitalized because of extensive bilateral pulmonary tuberculosis. After several weeks of tuberculostatic treatment--at first applied orally, then because of nausea and vomiting parenterally via a central venous catheter--he acutely developed nocturnal dyspnoea and symptoms of shock requiring artificial ventilation. Echocardiography demonstrated dilatation of the right ventricle and a large floating thrombus in the right atrium. During the examination complete displacement of the worm-like thrombus into the pulmonary artery was observed echocardiographically. Despite anticoagulation and immediate operation the patient died 2 days later of protracted shock. The thrombus was found to have been formed in the superior vena cava after placement of the central venous catheter.